Mechanism of Long Non-coding RNAs Action in leiomyoma

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Uterine leiomyoma (fibroids) are benign tumors afflicting a significant number of reproductive age women and without a known cause. Our laboratory has focused on understanding how miRNAs impact pro-inflammatory and pro-fibrotic pathways in leiomyoma, and identified two miRNAsmiR-200c and miR-29c which primarily target cell cycle, inflammation and extracellular matrix (ECM) component genes respectively as key in the development of leiomyoma. Our recent findings using next generation sequencing has demonstrated a whole host of non-coding RNAs including long non-coding RNAs (lncRNAs) are dysregulated in fibroids. LncRNAs can act as sponge for miRNAs and therefore may be a driver of gene dysregulation in leiomyomas. Based on the level of expression and functional association with tumorigenesis and tissue fibrosis we selected a group of lncRNAs to further characterize. Using QRT-PCR we detected marked overexpression of XIST and MIAT in leiomyomas. The sequences of these lncRNAs and preliminary data suggest they can act as miRNA sponges for miR-29c and miR-200c. Therefore, based on our published and preliminary data we hypothesize that leiomyoma as compared to myometrium display an altered expression of a number of lncRNAs that are transcriptionally regulated and independently or through miRNA-guided mechanisms regulate the expression of specific target genes functionally associated with cell cycle progression, inflammation, fibrosis and epigenetic modification all of which are central to leiomyoma pathogenesis. To test our core hypothesis we propose 3 specific aims. In Aim 1 we will build on our preliminary RNA sequencing study and provide a comprehensive profile of lncRNAs, miRNAs, with concurrent mRNAs expression in large sets of paired leiomyoma and myometrium from different race/ethnic groups derived from proliferative and secretory phase of the menstrual cycle. Through bioinformatic analysis we will identify lncRNAs subtypes, their chromosomal locations at loci often rearranged in leiomyoma and identify lncRNAs which could potentially act as miRNA sponges, or competing endogenous RNAs (ceRNAs). We will also determine if presence of MED12 mutations has any impact on the profiles of these ncRNAs. Aim 2 will address the regulation and function of MIAT and XIST in leiomyoma. Using RNA immunoprecipitation studies we will determine if these lncRNAs will interact with miR-29c and miR-200c known to be important in fibrogenesis. At the cellular level we will assess the role of ovarian steroids on their expression and through knockdown and knock-in strategies identify their effects on expression of ECM and cell cycle genes. To establish the clinical relevance and therapeutic potential of lncRNAs in Aim 3 we will determine the effect of altering the expression of XIST and MIAT which are highly overexpressed in fibroids on fibroid progression and/or establishment in a leiomyoma animal model. This translational proposal addresses a significant gap in knowledge in the pathogenesis of fibroids which is a priority area of investigation for NIH, and could have potential therapeutic applications for treatment of fibroids.